Journal of The Malacological Society of London Molluscan Studies Journal of Molluscan Studies (2013) 79: 372–377. doi:10.1093/mollus/eyt029 Advance Access publication date: 23 August 2013 RESEARCH NOTE EGG-HULL ULTRASTRUCTURE OF ISCHNOCHITON STRAMINEUS (SOWERBY, 1832), A SOUTH AMERICAN BROODING CHITON (CHITONINA: ISCHNOCHITONIDAE) Marı´a G. Liuzzi1 and Diego G. Zelaya2 1Divisio´n Invertebrados, Museo Argentino de Ciencias Naturales, CONICET, A´ngel Gallardo 470, Buenos Aires (C1405DJR), Argentina; and 2Departamento Biodiversidad, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, CONICET, Argentina Correspondence: M.G. Liuzzi; e-mail: [email protected] Reproductive aspects of the life history of chitons have been rela- Celebron˜a(¼Kidney) Island, Malvinas/Falkland Islands tively little studied in comparison with some morphological (518370S–578450W). Thirty-eight eggs were removed from the characters, such as valve morphology, girdle scales and radulae, pallial groove, dehydrated in an ascending ethanol series (from which have historically been considered of taxonomic value. In 70 to 100%), dried in an EMS 850 critical-point dryer using recent times, however, some authors have pointed out that char- liquid CO2, coated with gold-palladium (40–60%) and photo- acters related to reproduction, such as egg-hull morphology, are graphed with a Phillips XL-30 scanning electron microscope. also valuable for taxonomic purposes (Eernisse, 1988; Hodgson One juvenile was dehydrated and photographed in the same et al., 1988; Sirenko, 1993; Pashchenko & Drozdov, 1998; Okusu way as the eggs, but treated with hexamethyldisilazane and air et al., 2003; Buckland-Nicks, 2006, 2008; Sirenko, 2006a; dried instead of critical-point dried. The maximum length of the Buckland-Nicks & Schander, 2008; Buckland-Nicks & Reunov, egg plate and micropore diameter, two characters of taxonomic- 2009; Ituarte, Liuzzi & Centurio´n, 2010). Egg-hull morphology al value at suborder and family levels (Eernisse, 1988; Sirenko, varies not only among different suborders (i.e. Chitonina, with 1993; Pashchenko & Drozdov, 1998; Buckland-Nicks, 2008), spinose hull projections and narrow bases, vs Acanthochitonina, were measured, although the absolute values here provided with large hull cupules and wide bases), but also when compar- should be considered with caution owing to the long time that ing species with different modes of development (i.e. free- these specimens had been preserved in ethanol since collection. spawning vs brooding species) (Eernisse, 1988; Sirenko, 1993; Fifteen eggs were embedded in epoxy resin (Historesin Leicaw). Buckland-Nicks, 2008). Despite the valuable information pro- Sections (3.5–6 mm thick) were stained with haematoxylin- vided by this character, egg-hull morphology of southwestern eosin. Atlantic chitons remains poorly known. The only study dealing The two specimens containing eggs in the pallial groove were specifically with this issue is that by Ituarte et al. (2010) on collected in June 1921 and August 1955 (austral winter), while Plaxiphora aurata and Chaetopleura isabellei. the specimen bearing juveniles, with fully developed eight-valved Based on morphological characters, Sowerby (1832) described shells, was collected in December 1933 (austral summer). Eggs Ischnochiton stramineus (as Chiton stramineus) from Chiloe´Island (Fig. 1F) are packed in compact masses on both sides of the foot (Chile). Plate (1898) enlarged the description of this species (as (Fig. 1A, B), up to 1600 eggs in one of the specimens (Fig. 1A). Ischnochiton imitator (Smith), regarded as a synonym of I. strami- Egg hulls are formed of polygonal, perforated plates, about neus by Kaas & Van Belle, 1990), providing novel information 30 mm in width (Fig. 1D). Micropores (0.2–0.5 mm; Fig. 1D) are on its brooding behaviour and the general egg morphology randomly distributed over the entire surface of each plate, except (studied with light microscopy). More than a century later, for the area near the base of the projections (Fig. 1C). The per- Sirenko (2006b) reported for the second time the parental care iphery of the plates is raised, forming rounded, unperforated in this species. The present contribution provides additional ridges (Fig. 1C, D). Each plate shows a small subcentral eleva- information on the reproductive characteristics of I. stramineus, tion, which is the base of a hollow and elongated projection with focusing particularly on egg-hull ultrastructure. a spiral tip, the latter distally enlarged in a small ampoule The source of information for this study comes from three (Fig. 1C, E–G). These curled projections (numbering approxi- ethanol-preserved specimens (11.5 to 16.9 mm in length, brood- mately 204 per egg hull) interlock with those of other eggs and ing eggs or juveniles) in the collection of the Museo Argentino remain even in developing embryos (Fig. 1H). Seven fully meta- de Ciencias Naturales “Bernardino Rivadavia”: 1 specimen morphosed young, of about 6 mm length, were found in one of (MACN–In 12483) with eggs from Puerto Harris, Isla Dawson, the studied specimens (Fig. 1I). Our observations suggest that Magellan Strait (53850.00S–70823.10W); 1 specimen (MACN– I. stramineus eggs (or at least some of them) are retained in the In 22048) with juveniles from Puerto Olla, Isla Observatorio, pallial grooves until completely developed. Isla de los Estados Archipelago (54839.40S–64808.70W; The most frequent reproductive mode in chitons is the free 18–37 m); and 1 specimen (MACN–In 30695) with eggs from spawning of gametes followed by indirect development, while # The Author 2013. Published by Oxford University Press on behalf of The Malacological Society of London, all rights reserved RESEARCH NOTE Figure 1. Ischnochiton stramineus. A, C–H. Specimen from Malvinas/Falkland Islands (MACN–In 30695). B. Specimen from Magellan Strait (MACN–In 12483). I. Specimen brooded in the pallial groove of the specimen from Isla de los Estados Archipelago (MACN-In 22048). A, B. Reflex images. C–G, I. SEM images. H. Optical microscope image. A, B. Ventral view of adult specimens with compact mass of eggs stored in the pallial groove. C. Detail of a polygonal egg plate. D. Detail of micropores and junction between plates; arrow points to the rounded ridge. E. Detail of the spiral egg hull projections. F. General view of an egg. G. Detail of a hollow broken projection. H. Histological section of a developing embryo showing curled egg hulls. I. One of the seven juveniles fully metamorphosed. 373 RESEARCH NOTE Table 1. Brooding chiton species and shape of egg-hull processes. Order Suborder Species Brood Form of egg-hull Source (alphabetically processes arranged within suborders) Lepidopleurida Lepidopleurina Leptochiton Eggs and juveniles Unknown Sirenko & Schro¨dl, 2001; Sirenko, 2006b kerguelensis Lepidopleurida Lepidopleurina Leptochiton Eggs and juveniles Unknown Strack, 1987 algesirensis Lepidopleurida Lepidopleurina Hanleyella asiatica Eggs and juveniles [Spinose projections Sirenko, 1973; Sirenko, 1993 with curled tip] Chitonida Acanthochitonina Cyanoplax Eggs and Unknown Strack, 1987 (as Lepidochitona caboverdensis) caboverdensis embryos** Chitonida Acanthochitonina Cyanoplax caverna Eggs and Reduced cone-shaped Heath, 1905 (as Trachydermon raymondi); Eernisse, embryos** cupules 1988 (as Lepidochitona caverna) Chitonida Acanthochitonina Cyanoplax cinerea Eggs and Elongated cupules / Knorre, 1925 (fide Pearse, 1979 and Strack, 1987 embryos*,** short cupules1 as Lepidochitona cinereus); Matthews, 1956 (fide Pearse, 1979 as L. cinereus); Durfort et al., 1982 (as Trachydermon cinereus); Richter, 1986 (as L. cinerea); Sirenko, 1993 (as L. cinerea); Okusu et al., 2003 (as L. cinerea); Sirenko, 2006a (as L. cinerea) Chitonida Acanthochitonina Cyanoplax corrugata Eggs and Unknown Kowalevsky, 1883 (as Chiton polii); Strack, 1987 embryos** (as Lepidochitona corrugata) Chitonida Acanthochitonina Cyanoplax fernaldi Eggs and juveniles Cupules reduced to Eernisse, 1988 (as Lepidochitona fernaldi); flattened hexagonal Buckland-Nicks, 1993 (as L. fernaldi); Sirenko, 1993 plates (as L. fernaldi); Buckland-Nicks & Eernisse, 1993 (as L. fernaldi); Buckland-Nicks, 2008 Chitonida Acanthochitonina Cyanoplax Eggs and Unknown Scuderi, RUSSO & DELL’ANGELO, 2004 monterosatoi embryos** (as Lepidochitona monterosatoi) Chitonida Acanthochitonina Cyanoplax stroemfelti Eggs and Cone and cup-like Strack, 1987 (as Lepidochitona stroemfelti) embryos** cupules Chitonida Acanthochitonina Cyanoplax thomasi Eggs and juveniles Cupules reduced to Heath, 1905 (as Nuttallina thomasi); Thorpe flattened hexagonal (in Smith, 1966 as N. thomasi); Eernisse, 1988 plates (as Lepidochitona thomasi) Chitonida Acanthochitonina Hemiarthrum Eggs and juveniles Unknown Martens & Pfeffer, 1886; Simpson, 1977 setulosum Chitonida Acanthochitonina Placiphorella borealis Eggs [Reduced peripheral Sirenko, 1993 skirt-like processes] Chitonida Acanthochitonina Plaxiphora australis Eggs and juveniles Unknown Dell, 1963 (fide Creese, 1986 and Murdoch, 1982); Creese (in Strack, 1987) Chitonida Acanthochitonina Schizoplax brandtii Eggs and juveniles [spinose projections Kussakin, 1960 (fide Smith, 1966); Sirenko, 1993 with curled tip distally enlarged in a ampoule] Chitonida Chitonina Callistochiton Juveniles Unknown Smith, 1966 (as Lepidozona asthenes); Ferreira, 1979; asthenes Lindberg (in Pearse, 1979) Chitonida Chitonina Callistochiton leei Juveniles Unknown Ferreira, 1979 Chitonida Chitonina Callochiton crocinus